The present invention relates to a process for preparing alanine, especially L-alanine, by culturing a genetically modified microorganism that is able to produce alanine from a carbon source.
A process of this type is already known from Uhlenbusch et at., Appl. Environ. Microbiol. 57: 1360-1366 (1991). In this process, L-alanine is produced by culturing a microorganism (Zymomonas mobilis) that has been genetically modified in such a manner that it expresses an alanine dehydrogenase gene. The expressed alanine dehydrogenase converts the glucose in the medium, via conversion into pyruvate, into a mixture of alanine and ethanol, with a maximum alanine yield of 16%, calculated on the amount of glucose converted.
However, Zymomonas mobilis is not a food-grade organism. Nor is Zymomonas mobilis a homofermentative organism, since it forms one molecule of CO2 for each molecule of ethanol produced so that it forms 66% of ethanol and 33% of CO2, calculated on the carbon balance.
L-alanine is used in pharmaceutical and veterinary applications. For example, it is included, together with other amino acids, in preparations for parenteral administration as clinical preoperative and postoperative foods, as well as an animal feed supplement. Furthermore, alanine is used as a food additive on account of its sweet taste.
L-alanine is produced industrially by the decarboxylation of L-aspartate by means of immobilized cells or cell suspensions of Pseudomonas dacunhae. The L-alanine yield may, in this case, exceed 90%, starting from aspartic acid, but this is an expensive substrate.
D/L-alanine can also be prepared by chemical synthesis or by the direct fermentation of sugars. The microorganisms involved in this fermentation, such as Corynebacterium gelatinosium, Arthrobacter oxydans, Brevibacterium lactofermentum, Clostridium sp. and Pyrococcus furiosus, produce D/L-alanine with a maximum conversion rate of 50 to 60%, probably via catalysis by an endogenous alanine dehydrogenase.
EP 0 603 865 describes a process for the production of alanine, in which a microorganism of the genus Escherichia, Cornyebacterium or Brevibacterium, provided with a heterologous L-alanine dehydrogenase activity (for example from Arthrobacter), is used.
None of these microorganisms belongs to the lactic acid bacteria. However, a number of above-mentioned Brevibacterium-strainsxe2x80x94such as B.lactofermentum and B.flavumxe2x80x94are classified as among the xe2x80x9cfood gradexe2x80x9d microorganisms. However, this is because they are isolated from food products; they are not applied in the (fermentative) preparation of foodstuffs.
Finallyxe2x80x94as far as can be derived from the data given- according to Example 7 of EP 0 603 865 only a maximum conversion of 30% is reached, calculated on the amount of glucose in the medium.
EP 0 567 644 describes a process for the preparation of L-alanine by fermentation using an Arthrobacter-strain. In doing soxe2x80x94as far as can be derived from the data givenxe2x80x94according to Example 5 a maximal conversion of 30% is reached, calculated on the amount of glucose in the medium.
However, the Arthrobacter-strains used are not homo-fermentative and do not belong to the food grade or lactic acid bacteria. Furthermore, according to EP 0 603 865 page 5, lines 1-4 the use of Arthrobacter leads to contamination with xe2x80x9codor producing materialsxe2x80x9d.
J. Oerlgysson et al., Anthonie van Leeuwenhoek, 68 no.4, p. 273-280 (1995) describe the production of alanine using a Clostridium-strain. As far as can be derived from the data given the maximal conversion reached in this case is always less than 50%, calculated on the amount of glucose in the medium. Furthermore, Clostridium is homo-fermentative and does not belong to the food grade- or lactic acid bacteria. Also, on p. 279 it is mentioned that xe2x80x9cClostridium P2 produced almost no alanine from pyruvate, and, in fact, could not grow on pyruvatexe2x80x9d.
The use of E.coli-strains in the production of alanine is described in A. Galkin et al., Appl. Environ. Microbiology, 1997, p.4651-4656, and in Patent Abstracts of Japan, vol. 11, no.222 (C-435), corresponding to JP-A-62936196. However, E.coli is a hetero-fermentative organism (especially when cultured under minimum O2 conditions) and not a food grade- or lactic acid bacterium.
T. Ferain et al., J. of Bacteriology 1996, p. 5431-5437, describe a L.plantarum-strain (LDH), of which the two genes encoding the native lactate dehydrogenase are disrupted.
However, this reference does not describe the re-routing of the carbon/pyruvate metabolism in this organism towards the formation of alanine; therefore the LDHxe2x88x92-mutants can not be applied in the production of alanine.
The aim of the invention is to provide an improved process for the production of alanine, especially L-alanine.
It has now been found that a process of this type can be provided by replacing, in a suitable microorganism, the native metabolic conversion or conversions of sugars into their derivatives (such as lactate) by the formation of alanine, especially L-alanine.
The invention therefore relates to a process for preparing alanine, especially L-alanine, by culturing a genetically modified microorganism that is able to produce alanine from a suitable carbon source in the medium, characterized in that the microorganism used is one whose native sugar metabolism has been diverted to alanine formation, especially L-alanine formation, in such a manner that essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the carbon consumed from the medium is converted into alanine.
According to the invention preferably food grade- and/or lactic acid bacteria are applied and more preferably food grade strains of lactic acid bacteria. However, the invention is not limited to this:
when in the process of the invention a lactic acid bacterium is used (including the preferred food grade lactic acid bacteria), according to the invention essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the carbon consumed from the medium is converted into alanine;
when a food grade microorganism is used that does not belong to the lactic acid bacteria, according to the invention essentially more than 40%, preferably more than 60% and more preferably more than 75% of the carbon consumed from the medium is converted into alanine;
when a microorganism is used that is neither a food grade microorganism nor a lactic acid bacterium, according to the invention essentially more than 60%, preferably more than 75% of the carbon consumed from the medium is converted into alanine.
Furthermore, the microorganism used is preferably essentially homo fermentative.
In particular, according to the invention the native metabolic conversion or conversions of pyruvate (derived from the conversion of glucose or another suitable source of sugar in the medium) is/are diverted to alanine formation, in such a manner that essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the pyruvate formed being converted into alanine, on the basis of carbon in the pyruvate formed:
when in the process of the invention a lactic acid bacterium is used (including the preferably applied food grade lactic acid bacteria), according to the invention essentially more than 20%, preferably more than 40%, more preferably more than 60% and most preferably more than 75% of the pyruvate formed is converted into alanine;
when a food grade microorganism is used that does not belong to the lactic acid bacteria, according to the invention essentially more than 40%, preferably more than 60% and more preferably more than 75% of the pyruvate formed is converted into alanine;
when a microorganism is used that is neither a food grade microorganism nor a lactic acid bacterium, according to the invention essentially more than 60%, preferably more than 75% of the pyruvate formed is converted into alanine.
The process provides alanine to a high degree of purity and in a high yield, and with an appreciably greater efficiency of carbon utilization in comparison with known processes. In this process it is possible to obtain a re-routing of more than 80% and even more than 90% of the carbon introduced as sugar source into alanine, or even an essentially complete diversion ( greater than 99%).
The invention also provides, in a preferred embodiment, an improved stereospecificity for L-alanine in the end product, it being possible for a stereo-specificity of over 95% or even 99% or more to be achieved.
The invention further provides, in a special embodiment, a suitable technique for the in-situ preparation of alanine during the preparation of fermented foodstuffs or food preparations.
The carbon source used may be any metabolizable or fermentable carbon source whose native metabolism can be diverted to alanine formation, as described herein. This will generally be a carbon source that the microorganism natively converts into pyruvate, inter alia via glycolysis-based reactions. The carbon source is generally a conventional metabolizable and fermentable sugar source such as glucose, lactose (for lactic acid bacteria, see de Vos and Simons in xe2x80x9cGenetics and Biotechnology of Lactic Acid Bacteriaxe2x80x9d, Gasson and de Vos eds., pp. 52-106, Chapman and Hall, 1994), sucrose (for lactic acid bacteria, see Rauch and de Vos, 1992a, J. Bacteriol. 174: 1280-1287 and Rauch and de Vos, 1992b, Gene, 121: 55-61), maltose or starch (for lactic acid bacteria, see Asseldonk et al., 1993, Mol. Gen. Genet., 240: 428-434). Glucose, lactose and sucrose are generally preferred.
The degree of diversion of the sugar metabolism to alanine formation is expressed according to the invention on the basis of carbon (the carbon balance), as is customary in this field, i.e. as [the amount of carbon (atoms) in the alanine formed]/[the amount of carbon (atoms) in the sugar source consumed]xc3x97100%.
The amount of alanine formed (in grams or moles) per gram or mole of sugar consumed will depend on (the number of carbon atoms in) the sugar source. Therefore, on the basis of 100% conversion, 1 mol of glucose will give 2 mol of alanine, whilst 1 mol of sucrose or lactose will give 4 mol of alanine.
The re-routing of the native sugar metabolism to alanine formation can be brought about by conferring an (increased) alanine-producing activity on the microorganism and preferably at the same time disrupting or suppressing the native conversion or conversions of pyruvate.
This (increased) alanine-producing activity can be conferred by bringing one or more homologous or heterologous genes coding for an alanine-forming activity in the microorganism to (increased) expression.
These genes will in particular be (one or more) structural genes coding for an enzyme, known per se, that catalyzes the formation of alanine, especially L-alanine, particularly an enzyme that can produce alanine from a fermentable sugar source in the medium, as described above, or a metabolite thereof.
The enzyme preferably used is one that is able to produce alanine by converting pyruvate into alanine, especially an alanine dehydrogenase. The structural gene that codes for this can be derived from any suitable microorganism but is preferably an alanine dehydrogenase from a lactic acid bacterium or from a bacterium of the Bacillis genus, especially Bacillus sphaericus. In particular, an alanine dehydrogenase derived from a food-grade microorganism will be used. It is also possible to use the alanine dehydrogenase that is endogenous to the microorganism and which is, if required, brought to over-expression for this purpose by prior induction and/or under the control of a suitable homologous or heterologous promoter.
As an example of an alanine-dehydrogenase (gene) from a microorganism that does not belong to the food grade microorganisms or lactic acid bacteria, the L-alanine dehydrogenase from Arthrobacter can be mentioned, of which the cloning is described in EP 0 603 865.
The native conversion or conversions of the pyruvate metabolism can be suppressed by inhibiting or blocking the corresponding metabolic route, for example through the presence in the medium of reversibly or irreversibly inhibiting factors for the enzymes involved in this route, or by disrupting and/or suppressing the native expression of these enzymes.
However, it is preferred to use microorganisms that have been genetically manipulated in such a manner that they are deficient in the native conversion or conversions of pyruvate, for example because they express the corresponding enzymes in a greatly reduced or defective way or not at all, such mutants being well known to the experts.
One example is the lactate-dehydrogenase-deficient strain of L. lactis described by Platteeuw et al., 1995, Appl. Environ. Microbiol., 61: 3967-3971. In this article, the pyruvate metabolism of this strain is diverted to diacetyi production by the over-production of alfa-acetolactate synthetase (to the extent of approximately 80%, depending on the growth conditions). However, in this case other products are formed in addition to diacetyl, so that only a small portion of the carbon source consumed is converted into the required product (diacetyl), whilst the conversion carried out upsets the redox balance of the microorganism, which is a considerable disadvantage.
The microorganism used may also be any well-known microorganism, or a well-known mutant thereof, and both a homofermentative and a heterofermentative microorganism may be used.
Homofermentative microorganisms are understood to mean those which convert sugars via pyruvate essentially (i.e. to the extent of over 80%, calculated on the carbon balance) into a single metabolic product. This includes, for example, lactic acid bacteria such as L. lactis and S. cremoris, which convert pyruvate essentially completely into lactic acid/lactate. Reference is made to H. G. Schlegel: Allgemeine Microbiologie [General Microbiology], 5th ed., Thieme Verlag, 1981, especially pp. 255-295.
Other examples are Clostridia species such as C. acidi-urici and C. cylindro-sporum, and the homofermentative subspecies C. formicoaceticum and C. thermo-aceticum, which form acetate in a homofermentative manner from hydrogen equivalents (from substrate oxidation) and carbon dioxide according to the reaction scheme:
8[H-equivalents]+2CO2. . .  greater than CH3COOH+2H2O
However, these Clostridia species are generally not food-grade microorganisms.
Heterofermentative microorganisms are understood to mean those which transform the pyruvate into a number of principal conversion products. In the broadest sense, they include all the microorganisms that according to the above definition are not classed with the homofermentative microorganisms.
According to the present invention, the use of a homofermentative microorganism will generally be much preferred because in this case, replacing the conversion of pyruvate into the principal product derived from it by the conversion to alanine will already lead to an alanine production in a very good yield. For this purpose, furthermore, only one native route of the pyruvate metabolism has to be diverted, suppressed and/or disrupted. However, the use of heterofermentative microorganisms, in the case of which one or more of the principal native conversions of pyruvate is/are diverted to alanine production, is also within the scope of the present invention.
There are also some xe2x80x9csemi-homofermentativexe2x80x9d microorganisms which are known and which produce CO2 in addition to a single main product (organic molecule). Although these microorganisms cannot strictly be classified with the homofermentative organisms according to the above definition (formation of over 80% of a single metabolic product, determined on the basis of total carbon), they may be regarded according to the present invention as equivalents of purely homofermentative microorganisms whenever they produce over 80% of a single metabolic product, determined on the basis of the carbon balance, where CO2 is ignored. Another reason for this is that, when the metabolism of such an organism is diverted according to the invention, the final culture medium will essentially contain only alanine as the principal product, because the CO2 formed from the medium will be released into the surrounding air. However, the use of purely homofermentative organisms will still be preferred.
The microorganism used is preferably a lactic acid bacterium, such as L. lactis, L. bulgaricus, L. acidophilus, L. helveticus, S. cremoris or S. thermophilus, Lactobacillus plantarum, or another homofermentative microorganism that can utilize lactose, sucrose, glucose and/or pyruvate as substrate. For some applications, the use of food-grade microorganisms will be preferred.
The microorganism used is preferably deficient in activity or activities and/or enzymes that compete with the alanine-producing enzyme for the available substrate. For example, when the alanine-producing enzyme is an L-alanine dehydrogenase gene, the microorganism used is preferably one that is deficient in the enzyme lactate dehydrogenase, which can convert pyruvatexe2x80x94the substrate for L-alanine dehydrogenasexe2x80x94into lactate.
For the stereoselective production of L-alanine, the microorganism preferably used does not contain any appreciable alanine racemase activity either. For this, any native racemase activity can be suppressed in a known manner, for example by including in the medium some reversibly or irreversibly inhibiting factors, by disrupting and/or suppressing the native expression of the racemase activity, or by using microorganisms that have been genetically manipulated in such a manner that they express any native racemase activity in a greatly reduced or defective way or not at all. Such mutants will also be well known to the experts.
The one or more homologous or heterologous genes coding for the alanine-forming activity can be brought to (over-) expression in the microorganism used in a well-known manner, for example under the control of a conventional homologous or heterologous promoter that is active in the microorganism used.
For this purpose, the one or more structural genes are actively combined with the promoter, for example by introducing the coding gene in the right orientation and the right reading frame into a plasmid or another suitable vector, which already contains the promoter in question. The microorganism is then transformed in the usual way with the aid of the resulting recombinant DNA molecule, it being possible for the recombinant DNA to be either included in the bacterial genome or used as a separate plasmid in the cell. For all this, the well-known recombinant techniques, as described in Sambrook et al., xe2x80x9cMolecular Cloning: A Laboratory Manualxe2x80x9d, 2nd edition., vols. 1-3, Cold Spring Harbor (1989) may be used.
In principle, any suitable constitutive or inducible promoter that is active in the microorganism used may be used for this purpose. Examples of suitable constitutive promoters are the usp45 promoter (van Asseldonk et al., 1990, Gene, 95: 155-160), the nisR promoter (de Ruyter et al., 1997, J. Bacteriol., 178: 3434-3439), the pepN promoter (Tan et al., 1992, FEBS Lett., 306: 9-16) and the promoters mentioned in de Vos and Simons in xe2x80x9cGenetics and Biotechnology of Lactic Acid Bacteriaxe2x80x9d, Gasson and de Vos, eds., pp. 52-106, Chapman and Hall, 1994. Examples of suitable regulated promoters are the xc3x9631 middle promoter and ori-based expression system (O""Sullivan et al., 1996, Biotechnology, 14: 82-87), the xylA promoter (Lokman et al., 1994, Mol. Gen. Genet., 245: 117-125), and the repressor/operator xc3x96 rlt system (Nauta et al., 1996, Mol. Microbiol., 19: 1331-1341).
The promoter used is preferably such that the alanine-producing activity can be brought to increased expression and more preferably to regulated over-expression.
A highly suitable class of promotersxe2x80x94auto-inducible promoters as described in, for example, Kuipers et al., TIBTECH, 15: 135-140xe2x80x94will be described below.
An important alternative to the use of independently replicating recombinant structural units (plasmids) that code for alanine dehydrogenase is the insertion of the alanine dehydrogenase gene into the chromosomal DNA of the microorganism used. This can be done in a well-known way, for example by using suitable phage vectors, F-factor plasmids or transposons, or by conjugation with a suitable donor strain. Examples are the nisin sucrose conjugative transposon Tn5276 (Rauch and de Vos, J. Bacteriol., 174: 1280-1287), transposon Tn919 (Hill et al., 1985, FEMS Microbiol. Lett., 30: 115-119) and pGHOST integrative vectors (Magiun et al., 1992, J. Bacteriol., 174: 5633-5638).
The alanine dehydrogenase gene involved here is generally brought to expression under the control of an endogenous promoter in the bacterial DNA, although chromosomal insertion of the alanine dehydrogenase gene, combined with a suitable homologous or heterologous promoter (such as the promoters mentioned above) is also possible.
According to a particular embodiment of the invention, the structural gene coding for the native enzyme which can compete with the alanine dehydrogenase gene for the substrate (pyruvate), such as the gene coding for lactate dehydrogenase in lactic acid bacteria, is replaced in the chromosomal DNA by the alanine dehydrogenase gene, which can, if required, be brought under the control of the native promoter for the replaced gene.
The microorganisms transformed in this way, which can bring an alanine-producing activity to increased expressionxe2x80x94in comparison with the original/native microorganismxe2x80x94and which are at the same time preferably deficient in any competing native metabolism, especially in the native pyruvate metabolism, form another aspect of the invention.
The microorganism is then cultured under conditions ensuring the induction of the promoter and therefore the expression of the coding gene, after which the enzyme expressed is made to produce alanine from the constituents of the culture medium or its metabolites, especially glucose or another suitable fermentable carbon/sugar source. The culture medium can also contain all the conventional constituents of culture media, such as a suitable nitrogen source, together with trace elements and the like.
When the alanine-producing enzyme is an L-alanine dehydrogenase, the culture medium contains a source of ammonium ions in addition to a metabolizable carbon source or another pyruvate source. Ammonium ions are preferably present in the medium in a ratio of carbon source to ammonium ions of at least 1:3, calculated on the carbon consumed. In the case of glucose as the carbon source, for example, the ratio of glucose to ammonium is at least 1:2, while in the case of sucrose or lactose as the carbon source this ratio will be at least 1:4.
The expression of the alanine-producing enzyme and the formation of L-alanine can be controlled separately by the addition of the inducing factor for the promoter and the substrate for the alanine-producing enzyme, respectively. In this case, the expression and the L-alanine production can be induced/obtained either simultaneously or successively, as described in detail below.
The level of expression of the alanine dehydrogenase is not very important in itself, provided that enough alanine is produced. This will generally depend on the microorganism used. For example, after the suppression/disruption of the natively competing routes, the endogenous alanine-forming activity present in the microorganism used may already be sufficient to produce enough alanine. When homologous or heterologous alanine dehydrogenase is brought to intensified expression, this enzyme can constitute up to 30-40% or more of the cellular protein.
The formation of L-alanine can, furthermore, be carried out in a well-known manner by using conventional microbiological techniques and reactors for culturing/treating the microorganism used or processes similar to these. For example, batchwise or continuous processes can be used.
It is also possible according to the invention to use the microorganisms in an immobilized form on a suitable carrier, for example in an appropriate cell reactor or in a column.
However, the use of intact cells of the microorganism will generally be preferred according to the invention over the use of cell extracts such as cell-free extracts.
The alanine formed can then be isolated from the fermentation medium and possibly purified further by a conventional method.
The process according to the invention gives alanine to a high degree of purity ( greater than 90%, preferably  greater than 99%), the efficiency of glucose consumption also being high ( greater than 75%). Especially when any native racemase activity present is suppressed as described above, the process also gives a high level of stereo-specificity, an end product being obtained that contains more than 80%, especially more than 95% or even 100% of the L-isomer (calculated on the total amount of alanine) (total stereospecific production).
In further aspects, the invention relates to the alanine obtained according to the process described here, together with preparations, especially foodstuffs and/or health-promoting preparations, as well as additives therefor, which contain alanine produced according to the process. Furthermore, the invention may also be used for the large-scale production of the alanine-producing enzyme, as described above.
Finally, in a further aspect the invention relates to a very convenient system for controlling the expression of the alanine-producing activity, especially when the process according to the invention is carried out in lactic acid bacteria.
This aspect comprises the use of an xe2x80x9cauto-induciblexe2x80x9d promoter, which is understood to mean one that can be induced by the expression product of the coding gene which is natively controlled by this promoter, including any post-translation modified expression product, as well as analogues and derivatives of such expression products.
This aspect of the invention therefore relates to a process for preparing alanine, especially L-alanine, by culturing an alanine-producing genetically modified microorganism, in which the microorganism used is one that contains a gene coding for an alanine-producing enzyme under the control of an auto-inducible promoter that is active in the microorganism, this promoter being derived from a gene cluster that codes for an antimicrobial peptide or a similar signal protein.
According to this aspect, auto-inducible promoters that natively control the expression of gene clusters which code for antimicrobial peptides are used in particular. Such promoters can be induced by the antimicrobial protein itself, as well as by its analogues and derivatives, generally via a two-component signal transduction system.
Non-limiting examples are the promoters from the bacteriocin gene cluster of Carnobacterium piscicola, the Sakaricin gene cluster from Lactobacillus sake, as well as (presumably) the Subtilin gene cluster from Bacillus subtilis. According to the invention these also include promoters for similar, non-bactericidal signal proteins involved in the quorum sensing process in microorganisms, such as the promoter for Plantaricin A from the bacteriocin gene cluster of Lactobacillus plantarum. Reference is made to Kleerebezem et at., Molecular Microbiology (1997) 24 (5), 895-904 and the references mentioned therein.
The preferred promoters used are the auto-inducible promoters from the nisin gene cluster of L. lactis, especially the nisA and nisF promoters, as described in the Applicant""s European Application 0,712,935 (nis A) and also in De Ruyter et al., Appl. Environ. Microbiol., 62: 3662-3667 (1996); Kuipers et al., Tibtech, April 1997 (Vol. 15): 135-140, and the references given therein (nisA and nisF). These promoters have already been used for the controlled (over-) expression of homologous and heterologous proteins (such as the gusA reporter gene from E.coli, pepN, and the lytic genes lytH and lytA) in lactic acid bacteria and for the nisin-induced expression in heterologous hosts such as Lactobacillus helveticus and Leuconostoc lactis. According to the invention, this auto-inducible promoter system is advantageously used for the controlled (over-) expression of the alanine-producing activity, i.e. of the alanine-producing enzyme.
The gene that codes for the alanine dehydrogenase can be brought under the control of the nisin promoter in the manner described above by being actively combined with this promoter. For example, the coding gene can be introduced, in the correct reading frame, into a plasmid or vector that already contains an auto-inducible promoter, essentially in a similar way to the processes described in EP-A-0,712,935 and in the paper by De Ruyter et al. mentioned above.
Therefore, a further aspect of the invention relates to recombinant DNA molecules or sequences that comprise one or more genes coding for an alanine-producing activity, especially for an alanine-producing enzyme, such as L-alanine dehydrogenase that has been brought under the control of an auto-inducible promoter, especially a promoter from the nisin gene cluster as described above.
The recombinant DNA molecule is preferably in the form of a vector, such as a plasmid, which can at the same time also contain selectable markers and other well-known vector components. The preferred plasmids used are described below in more detail.
The microorganisms can be transformed with these vectors according to the invention in a well-known manner, for example as described in EP-A-0,712,935 and in the paper by De Ruyter et al. mentioned above.
The microorganisms transformed in this wayxe2x80x94which constitute a further aspect of the inventionxe2x80x94can be used for the production of L-alanine by being cultured in a medium that contains at least one inducing factor for the auto-inducible promoter in an inducing concentration. This ensures the expression of the alanine-producing activity, which can then be used for forming L-alanine from a suitable substrate that can be included in the medium.
Suitable inducing factors are notably (but not necessarily) the expression products of the gene/genes that is/are natively controlled by the promoter used, as well as analogues and derivatives thereof. They are generally antimicrobial proteins or similar xe2x80x9cpeptide pheromonesxe2x80x9d, such asxe2x80x94for the nis promotersxe2x80x94nisin A, nisin Z and analogues/derivatives thereof, as well as mutants, variants and/or fragments thereof.
The induction can also be carried out as described in EP-A-0,712,935 and in the above paper by De Ruyter et al., or in a similar way. This preferably ensures increased expression or over-expression of the desired L-alanine-producing activity in comparison with the native microorganism.
When an auto-inducible promoter is used, the cells of the microorganism used preferably contain (the genes for) a signal transduction system, which induces the promoter in response to the inducing factor present outside the cell. This system will often consist of two components (i.e. a sensor protein and a response regulator) as in the known nisK and nisR genes/proteins from the nisin gene cluster. This signal transduction system may even be present natively in the microorganism used, or be conferred on the microorganism by a suitable expression of heterologous genes. The preferred signal transduction system used is one that is natively involved in the control of the promoter used. Reference is made to the above paper by Kleerebezem et al. and the references mentioned therein.
The use of the auto-inducible promoter system according to the invention further ensures all the well-known advantages of this system, as described in EP-A-0,712,935. For example, it is possible to induce the expression of the alanine-producing activity at any desired moment during the fermentation and this expression varies essentially linearly with the concentration of the inducing factor in the medium (positive induction).
Furthermore, the preferred inducing factors used (nisin A, nisin Z and their analogues/derivatives) are acceptable for use in foodstuffs, because they are formed natively in the fermentation of dairy products by lactic acid bacteria. As a result, the invention may also be advantageously used in situ for preparing preferably fermented foodstuffs, for example for the controlled in-situ production of alanine as a sweetener in dairy products, especially in cheese or in fermented dairy products such as yoghurt. A further advantage here is that the alanine production can be induced without having to make any substantial changes to the nutrient (medium).
In this case, the microorganism can be used in combination with the common acidifying microorganisms, it being possible for the microorganisms according to the invention also to be derived from these common acidifying gents.